Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung

This study investigates how specific members of the lung microbiome influence the early immune response to infection. Prevotella species are a major component of the endogenous airway microbiota. Increased abundance of Prevotella melaninogenica correlates with reduced infection with the bacterial pathogen Streptococcus pneumoniae, indicating a potentially beneficial role. Here, we show that P. melaninogenica enhances protection against S. pneumoniae, resulting in rapid pathogen clearance from the lung and improved survival in a mouse lung co-infection model. This response requires recognition of P. melaninogenica lipoproteins by toll-like receptor (TLR)2, the induction of TNFα, and neutrophils, as the loss of any of these factors abrogates Prevotella-induced protection. Improved clearance of S. pneumoniae is associated with increased serine protease-mediated killing by lung neutrophils and restraint of P. melaninogenica-induced inflammation by IL-10 in co-infected mice. Together, these findings highlight innate immune priming by airway Prevotella as an important protective feature in the respiratory tract.

A clear point is made on associations between the presence of Prevotella ssp. and the induction of a milder inflammation (sub-clinical with neutrophil involvement) in the context of different pathogenic infections. Microbiome analyses have indicated that increased abundance of P. melaninogenica correlates with reduced infections.
Using a mouse co-infection model, the presented data show that P. melaninogenica induced an innate immune that resulted in some immune protection against S. pneumoniae infection through recognition via TLR2, induction of TNF , and neutrophil activation. Increased clearance of S. pneumoniae from the lung was due to increased serine protease-mediated killing resulting in improved survival.
The results presented here are highlighting the interactions od co-infections in the activation of the immune system. While a suggestion can be made regarding the importance of the whole microbiome in this process, as the study focused on a co-infection model consisting of two bacterial species, this should be made clear in the abstract and the conclusion.
This would take nothing away from the importance of the data but highlights that with our current methods we are not yet able to investigate the whole microbiome interactions in this manner.
Overall, the presented work is highly relevant to respiratory health and emphasis the important of co-infections (the microbiome?). Experiments are thoroughly performed, however, there are too many graphs (some show duplicate results). The manuscript would also benefit from some cleared and shorter description of the results.

Methods:
Various bacterial strains were used throughout the study. Most of them were grown in their specific medium or broth. Would the medium the strain was propagated in have any effect on its virulence? Please comment.
Please comment and add into the manuscript.
Prevotella aspiration was modelled in mice by instilling P. melaninogenica intratracheally (i.t.) prior to challenge with S. pneumoniae. The overall rationale for this was that the murine airway microbiome contains both Prevotella and Streptococcus species, but P. melaninogenica and S. pneumoniae are not resident members.
While the model allows for the addition of the required species to the existing lung microbiome, how does such addition change the whole microbiome (not only the response to each other, inflammatory immune responses, or bacterial clearance)? Can the overall effect be attributed to the interaction of P. melaninogenica with S. pneumoniae or with the overall change in the microbiome?
Please comment and add the information to the manuscript.
Statistical analysis: Experiments are performed at n=4-5 in each experiment.
Although the plotted data in the dot plot look as if they are normally distributed, the authors should consider non-parametric tests and descriptive measures for the small numbers investigated.

Results:
Pre-exposure (instillation) of heat-killed P. melaninogenica increased survival following a lethal dose of S. pneumoniae (fig 1b), however, when equivalent doses of P. melaninogenica and of S. pneumoniae were used only heat killed bacteria mediated the protection. "Heat-killed P. melaninogenica was protective against both serotype 2 S. pneumoniae, which spread systemically by 24 hours, and serotype 3 S. pneumoniae, which was restricted to the lung (Fig. 1d- Early clearance of a sublethal dose of S. pneumoniae in mice exposed to P. melaninogenica correlated with lower burdens in the lung 10 three days later (Supplementary Fig. 1a) In subsequent experiments only heat-killed P. melaninogenica was used. Importantly, heat killed E. coli did not mediate protection (Fig. 1f). However, as both bacteria are Gram negative species and TLR4 agonists, it appears that not all Gram-negative bacteria have a similar effect Analyses of serum cytokines indicated that the protective effect heat-killed P. melaninogenica was mediated by the induction of IL-10 and a reduction of TNF , differences in cytokines only seen in P. melaninogenica exposures.
What is the difference between Fig. 1g ("… less TNF and more IL-10 at 24 hours post-S. pneumoniae infection compared to those exposed to E. coli HK or E. coli LPS …") and Fig. 2b ("Mice exposed to P. melaninogenica also had increased systemic TNF and IL-10 compared to mice treated with PBS or E. coli LPS"). Can these graphs be combined?
Further analyses of BAL fluid showed a panel of innate immune cytokines (e.g., increase in IL-1 , IFN , MCP1, IL6, MIP-2) and the recruitment of neutrophils to the lungs after P. melaninogenica exposure (Ly6G+ cells). In contrast to E. Coli, P. melaninogenica also showed a stronger activation of the neutrophils (Ly6G+ / TNF + cells).
This overall suggests a ''selective' effect of P. melaninogenica on neutrophils, which potentially mediates the immune protection. When Ly6G+ cells or TNF from Ly6G+ cells are deleted, the protective effect on bacterial burden ceases.
Investigations into the host and bacterial requirements for P. melaninogenica-induced neutrophil TNF production showed the importance of TLR2 signalling for this response (inhibition of TLRs). Further, TNF induction was significantly reduced when heat-killed P. melaninogenica was treated with lipase to digest bacterial lipoproteins in the cell membrane, which are bacterial TLR2 ligands.
However, of interest is that additional factors appear to be required to mediate the protective TLR2lipoprotein signalling effect as neither Pam3SK4 (TLR2 agonist) nor purified P. melaninogenica lipoproteins reduced S. pneumoniae burdens in the lung (Fig. 4f).
In the co-infection model, neutrophil recruitment was lost in P. melaninogenica-exposed TLR2 -/mice infected with S. pneumoniae, while the recruitment of inflammatory monocytes was maintained. This is an important finding that should be added to the figures.
The effect of IL10 in the regulation of protective effects of TNF Using purified neutrophils, P. melaninogenica induced the secretion of IL-10 in a dose-dependent manner, which could regulate TNF production. Compared to the single infections, co-infection of bone marrow derived neutrophils with P. melaninogenica and S. pneumoniae shows significantly reduced TNF release. This is also shown as a systemic response in Prevotella-exposed mice. This is not in Fig. 1g, but in fig 7? Additionally, BALF cytokines TNF , IL-6, IL-1 , IFN and IFN were also reduced in co-infections. This was observed 48h after co-infections, suggesting that a regulator such as IL-10 must be induced, transcribed etc. before an effect can be seen on protein levels.
Additional effect of IL-10 derived from other immune cells.

Detailed comments
Line 5, page 2: "Prevotella are frequently [identified / found] among the top three most abundant bacteria detected in the oral cavity and lungs of healthy adults" -add verb Line 26, page 5 ff. "… which like S. pneumoniae are gram-positive." Please refer to Gram staining as Gram-positive or Gram-negative, acknowledging Dr Gram, the Danish bacteriologist who developed the technique in 1884. (https://www.newscientist.com/article/2216418-hans-christian-gram-thebiologist-who-helped-investigate-bacteria/). Please correct throughout.
Line 12, Page 21: " … with analytes were detected on the 14 LSR Fortessa X-20 in the ImmunoMicro Flow Cytometry Shared Resource Laboratory …" Omit 'were' Please check for typographical and grammatical errors throughout.

Response to Referees
April 28 th , 2022 Manuscript: NCOMMS-21-49627A Title: Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung Summary of Changes: We thank the reviewers for their time and critical analysis of our manuscript. Our revised manuscript includes extensive new data to address reviewer comments. Among these new data, we have included a new Figure and Supplementary Figure  [Fig. 4, Supplementary Fig. 4] focused on the role of the endogenous microbiome in P. melaninogenica-mediated protection against S. pneumoniae infection. We find that live P. melaninogenica is protective against S. pneumoniae in both antibiotic treated and in Germ-free mice, suggesting that exposure to P. melaninogenica is sufficient to enhance S. pneumoniae clearance without the participation of the endogenous microbiota [ Fig. 4a-b]. In Germ-free mice, we confirm that P. melaninogenica induces neutrophil recruitment and activation in the lungs of infected mice [ Fig. 4c], similar to mice with an intact microbiome. We also address whether other airway Prevotella mediate a similar protective effect against S. pneumoniae infection in a new Figure [Fig. 9]. We find that exposure to several other live airway Prevotella isolates, including another strain of P. melaninogenica (D18), P. buccae, P. tannerae, and P. nanceiensis, mediates rapid clearance of S. pneumoniae from the lung, similar to the effect of P. melaninogenica strain 25845 used throughout the original study. In contrast, the major periodontal pathogen P. intermedia does not improve protection against S. pneumoniae [ Fig.  9a]. In addition, we find that only Prevotella species which are protective against S. pneumoniae induce neutrophil secretion of TNF-alpha and IL-10 in a TLR2-dependent manner [ Fig. 9b]. Together, these data are consistent with an important role for TLR2-dependent neutrophil activation in Prevotella-mediated protection against S. pneumoniae. Additional new data [ Fig.  5f, Supplementary Fig. 2h, Supplementary Fig. 6c, Supplementary Fig. 7a, f] and highlighted edits to the manuscript text are described in the point by point responses below. We believe these new data prompted by reviewer input significantly improve the strength of our revised manuscript.

Reviewer #1 (Remarks to Author):
This manuscript, "Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of Streptococcus pneumoniae from the lung", provides an interesting addition to how resident microbes in the airway help protect against pathogenic microbes. It details how Prevotella melaninogenica (Pm) protects against Streptococcus pneumoniae (Sp) lung infection. Overall, I found the study to be thorough and well controlled. I think it adds several important findings to the field. I have the following points: 1) There are a few additional controls which would support the authors' mechanism. One confounder not addressed here is the indirect effect of Pm inoculation on microbial communities already resident in mice. Could the effect of Pm be indirect through its modulation of abundance of other organisms already resident in the upper airway or lung? This could be address by using antibiotic treatment in their existing models.
We agree that the potential influence of the endogenous microbiome on the protective effect mediated by P. melaninogenica is an important question. To address this, we conducted new experiments using live P. melaninogenica in both antibiotic-treated and Germ-free mice. In both cases, live P. melaninogenica significantly enhanced protection against S. pneumoniae lung infection as shown by improved pneumococcal clearance from the lung [ Fig. 4a-b]. We also confirmed that P. melaninogenica-mediated protection in Germ-free mice was associated with increased neutrophil recruitment and neutrophil expression of TNF-alpha by flow cytometry [Fig.  4c]. These results suggest that P. melaninogenica is sufficient to improve protection against S. pneumoniae lung infection in the absence of the endogenous microbiome.
2) Could any of the effects be driven from the gut? More specifically, are any of the Pm inoculated via the different routes used in this study getting to the gut and signalling from there?
A recent study published using the same method of i.t. instillation (from the lab in which corresponding author Dr. Clark was trained in this method) demonstrated using Trypan Blue dye that liquid injected i.t. doesn't reach the stomach, as dye was not detected in stomach tissue, unlike mice injected by oral gavage where the dye was clearly visible in the stomach [Bortell et al 2021, PMID:33878120]. This suggests that the large majority of the P. melaninogenica inoculated directly into the lung (i.t.) is not getting into the gut. While it is unclear whether the live P. melaninogenica instilled into the lungs are capable of spreading systemically after inoculation, it is unlikely that the heat-killed preparations of P. melaninogenica spread beyond the lung, given the Trypan Blue data. In contrast, P. melaninogenica inoculated intranasally may reach the gut, as a portion of this inoculum is likely swallowed by the animal. While intranasal inoculation was not our primary model, we note that the phenotype is similar to i.t. inoculation of heat-killed P. melaninogenica [ Supplementary Fig. 1b], suggesting that any priming due to bacteria reaching the gut has a limited impact in this setting.
3) Does this effect extend to other Prevotella? The authors only tested a very limited repertoire of other commensals.
We agree that the extent to which this protective mechanism is a conserved feature of airway Prevotella species is an interesting and important question. To address this, we conducted new experiments with the following additional live airway Prevotella isolates: another strain of P. melaninogenica (strain D18), Prevotella buccae, Prevotella tannerae, Prevotella nanceiensis, and P. intermedia. Among these, all except P. intermedia were protective against S. pneumoniae infection [ Fig. 9a]. P. intermedia is a major periodontal pathogen, and previously reported to enhance susceptibility to type 4 S. pneumoniae [Nagaoka et al 2014, PMID: 24478074]. These results indicate that several airway Prevotella species are capable of enhancing protection against S. pneumoniae, while more pathogenic species such as P. intermedia are not protective. To further investigate how these different Prevotella species activate neutrophils, we stimulated WT versus Tlr2 -/neutrophils with heat-killed preparations of each Prevotella isolate. We found that all protective Prevotella strains induced neutrophil secretion of TNF-alpha and IL-10 in a TLR2-dependent manner [Fig 9b]. In contrast, P. intermedia activated neutrophils in a TLR2-independent manner. These data build on findings presented in the original manuscript highlighting an important role for TLR2-dependent neutrophil activation, suggesting that activation of this innate immune pathway is a conserved feature among protective airway Prevotella species.
4) The analysis of the TNF/neutrophil axis is good. I think it's important to understand what the TNF levels in the lung are during neutrophil depletion. This would help to understand whether neutrophils are the sole lung TNF source or not.
To address this question, we depleted neutrophils in P. melaninogenica-exposed mice and compared BAL levels of TNF-alpha to those in Prevotella-exposed mice treated with isotype control antibody and naïve controls. Neutrophil depletion significantly reduced total TNF-alpha in the BAL of mice exposed to P. melaninogenica [ Supplementary Fig. 2h]. However, there is still some TNF-alpha in the BAL of neutrophil-depleted above that detected in naïve animals, indicating that other cellular sources are responsible for the remaining TNF-alpha induced by P. melaninogenica.

5)
In the IL-10 KO model, if TNF is neutralized does this rescue the defect caused by IL-10 loss?
The role of TNF-alpha in Il10-/-mice was investigated by TNF-alpha neutralization, as suggested. We find that TNF-alpha neutralization had no impact on S. pneumoniae burdens 24 h post-infection in Il10-/-mice [ Supplementary Fig. 7f]. These data indicate either that TNFalpha neutralization alone was not sufficient to restrain inflammation in these mice, or that too much TNF-alpha abrogation limited the protective immune activation phenotype (which requires TNF-alpha). Regardless, in the absence of IL-10, the modulation of TNF-alpha alone is not sufficient to restore protection. The critical protective versus tissue-damaging responses regulated by IL-10 following exposure to P. melaninogenica is an area of ongoing investigation.

6)
In the experiments where synthetic TLR2 ligands are administered to mice, they fail to promote Sp clearance promoting the authors to suggest TLR2 is necessary but not sufficient. However, could this be simply a dosing issue? Has a comparable amount of synthetic TLR2 ligand been added to the level of TLR2 agonists produced/displayed by Pm?
To address this question, we first determined that the total protein content of the HK P. mel. preparations injected per mouse was 41.9 μg by BCA Protein analysis. Using this value as a reference point for the highest potential amount of TLR2 ligand present, we titrated administration of Pam3SK4, this time using doses of 25-50 μg per mouse. Synthetic TLR2 ligand at the highest dose still failed to induce protection against S. pneumoniae [ Fig. 5f]. These data support the conclusion that TLR2 agonists alone are not sufficient to mediate protection against S. pneumoniae.

7) Are the serine protease inhibitors directly toxic to Sp?
We found that the one-hour exposure to the serine protease inhibitor cocktail used in these studies had no impact on S. pneumoniae growth, as measured by CFU/mL [ Supplementary  Fig. 6c]. 8) One of the most striking findings is that it looks like TLR2 is not important for direct recognition of Sp. Care has to be taken with the TLR2-/-mice because direct recognition of Sp could be lost (in addition to recognition of Pm). As a minimum, there must be some statistical comparison in the data shown that confirms, at the point of infection shown in most of the data (24h) TLR2 is not important for Sp defence and clearance.
Analysis of pooled CFUs from three independent experiments comparing WT vs Tlr2-/-mice at 24 hpi (n= 9 mice/group) confirms there is no difference in burdens of S. pneumoniae [ Fig. 6c Corrected as recommended.

Reviewer #2 (Remarks to Author):
Review Horn et al -Nat Communications Jan 2022 Airway Prevotella promote TLR2-dependent neutrophil activation and rapid clearance of 5 Streptococcus pneumoniae from the lung Kadi J. Horn, Melissa A. Schopper, Sarah E. Clark* This study investigates the potential interaction of Prevotella melaninogenica, one of the most commonly isolated species of the lung microbiome, with the bacterial pathogen Streptococcus pneumoniae, the most common cause of community-acquired pneumonia. A clear point is made on associations between the presence of Prevotella ssp. and the induction of a milder inflammation (sub-clinical with neutrophil involvement) in the context of different pathogenic infections. Microbiome analyses have indicated that increased abundance of P. melaninogenica correlates with reduced infections. Using a mouse co-infection model, the presented data show that P. melaninogenica induced an innate immune that resulted in some immune protection against S. pneumoniae infection through recognition via TLR2, induction of TNFα, and neutrophil activation. Increased clearance of S. pneumoniae from the lung was due to increased serine protease-mediated killing resulting in improved survival. The results presented here are highlighting the interactions od co-infections in the activation of the immune system. While a suggestion can be made regarding the importance of the whole microbiome in this process, as the study focused on a co-infection model consisting of two bacterial species, this should be made clear in the abstract and the conclusion. This would take nothing away from the importance of the data but highlights that with our current methods we are not yet able to investigate the whole microbiome interactions in this manner.
We thank the reviewer for these comments. While our revised manuscript expands the number of airway Prevotella species associated with increased protection against S. pneumoniae in our model, we agree that the focus remains on specific members of the airway microbiome, rather than the microbiome as a whole. As suggested, we clarified in the abstract and conclusion sections that our studies indicate airway Prevotella species, specifically, as important members of the respiratory tract microbiome for enhanced protection against S. pneumoniae infection in the lung [pg 1, line 25, pg 4, lines 7-10, pg 18, lines 10-13, and pg 19, line 2].
Overall, the presented work is highly relevant to respiratory health and emphasis the important of co-infections (the microbiome?). Experiments are thoroughly performed, however, there are too many graphs (some show duplicate results). The manuscript would also benefit from some cleared and shorter description of the results.
We carefully revised the results section to improve the clarity and brevity of our analyses. Graphs showing information similar to that already presented elsewhere have been moved to Supplementary figures. We note that all such graphs contain data from independent experiments, confirming foundational observations made throughout the manuscript. All flow cytometry data are now presented in a more simplified manner, with graphs showing total cell numbers which were next to similar graphs of cell percentages moved to Supplementary figures. We thank the reviewer for this suggestion, as these changes have improved the clarity of the main Figures.

Methods:
Various bacterial strains were used throughout the study. Most of them were grown in their specific medium or broth. Would the medium the strain was propagated in have any effect on its virulence? Please comment.
We are unaware of any media-dependent impact on virulence for the strains used in this study. We have grown S. pneumoniae in several different types of nutrient broth (e.g., Brain Heart Infusion with or without 1% Tween80, Todd Hewitt Broth with Yeast Extract) with similar results. For example, 24 h lung burdens from S. pneumoniae grown in Brain Heart Infusion with 1% Tween80 versus Todd Hewitt Broth with Yeast Extract are the same. Some, but not all, of the other bacterial strains used in these studies were grown in one of these media types (e.g., Corynebacterium species and S. salivarius). E. coli and Prevotella isolates were only grown using one type of media, which differed from that used for S. pneumoniae, however we report similar results between live and inactivated bacteria for both of these strains, suggesting a minimal impact of media on strain virulence. Of note, all bacteria prepared for inoculation into mice were resuspended in PBS, rather than inoculated in the original growth media, which also serves to minimize differences due to growth media.
Both of the mutant mouse strains used in these studies (Tlr2-/-and Il10-/-) are on the C57BL/6J genetic background, which is the same as the WT strain [clarified in Methods, pg 19, lines 18-19]. The nomenclature for these strains refers to the original gene disruptions, which were generated using strain 129 embryonic stem cells injected into C57BL/6J blastocysts. Heterozygotes were then backcrossed to C57BL/6J for at least 10 generations, maintained at the Jackson Laboratory from which they were purchased for this study.
Prevotella aspiration was modelled in mice by instilling P. melaninogenica intratracheally (i.t.) prior to challenge with S. pneumoniae. The overall rationale for this was that the murine airway microbiome contains both Prevotella and Streptococcus species, but P. melaninogenica and S. pneumoniae are not resident members. While the model allows for the addition of the required species to the existing lung microbiome, how does such addition change the whole microbiome (not only the response to each other, inflammatory immune responses, or bacterial clearance)? Can the overall effect be attributed to the interaction of P. melaninogenica with S. pneumoniae or with the overall change in the microbiome? Please comment and add the information to the manuscript.

From response above to Reviewer #1:
We agree that the potential influence of the endogenous microbiome on the protective effect mediated by P. melaninogenica is an important question. To address this, we conducted new experiments using live P. melaninogenica in both antibiotic-treated and Germ-free mice. In both cases, live P. melaninogenica significantly enhanced protection against S. pneumoniae lung infection as shown by improved pneumococcal clearance from the lung [ Fig. 4a-b]. We also confirmed that P. melaninogenica-mediated protection in Germ-free mice was associated with increased neutrophil recruitment and neutrophil expression of TNF-alpha by flow cytometry [Fig.  4c]. These results suggest that P. melaninogenica is sufficient to improve protection against S. pneumoniae lung infection in the absence of the endogenous microbiome.
Statistical analysis: Experiments are performed at n=4-5 in each experiment. Although the plotted data in the dot plot look as if they are normally distributed, the authors should consider non-parametric tests and descriptive measures for the small numbers investigated.
All data which passed the Shapiro-Wilk normality test were analyzed using parametric tests (ANOVA, t test), while all data which failed this test of normality were analyzed using nonparametric tests (Mann-Whitney U test, Kruskal-Wallis test). Data from infection experiments were pooled rather than showing representative data for Fig. 3b, Fig. 3e, Fig. 4 and Fig. 6c, for n=9-11 mice/group, though these data were not normally distributed due to the CFU limit of detection (relevant for P. melaninogenica treated groups) and were analyzed using nonparametric tests regardless. We acknowledge that the flow cytometry data from representative experiments with n=4-5 mice/group which passed the Shapiro-Wilk normality test could alternatively be analyzed using non-parametric tests and descriptive measures. To address this, we included non-parametric analyses for such data in Fig. 2 and Fig. 6  Results: Pre-exposure (instillation) of heat-killed P. melaninogenica increased survival following a lethal dose of S. pneumoniae (fig 1b), however, when equivalent doses of P. melaninogenica and of S. pneumoniae were used only heat killed bacteria mediated the protection. "Heat-killed P. melaninogenica was protective against both serotype 2 S. pneumoniae, which spread systemically by 24 hours, and serotype 3 S. pneumoniae, which was restricted to the lung (Fig. 1d-e)"  Early clearance of a sublethal dose of S. pneumoniae in mice exposed to P. melaninogenica correlated with lower burdens in the lung 10 three days later ( Supplementary Fig. 1a) In subsequent experiments only heat-killed P. melaninogenica was used. Importantly, heat killed E. coli did not mediate protection (Fig. 1f). However, as both bacteria are Gram negative species and TLR4 agonists, it appears that not all Gram-negative bacteria have a similar effect Analyses of serum cytokines indicated that the protective effect heat-killed P. melaninogenica was mediated by the induction of IL-10 and a reduction of TNFα, differences in cytokines only seen in P. melaninogenica exposures. What is the difference between Fig. 1g ("… less TNFα and more IL-10 at 24 hours post-S. pneumoniae infection compared to those exposed to E. coli HK or E. coli LPS …") and Fig. 2b ("Mice exposed to P. melaninogenica also had increased systemic TNFα and IL-10 compared to mice treated with PBS or E. coli LPS"). Can these graphs be combined? Data in Fig. 1g [now Fig. 1e] are from mice pre-exposed to P. melaninogenica and infected with S. pneumoniae, while data in Fig. 2b are from mice exposed to P. mel. HK or E. coli LPS alone (no S. pneumoniae infection). Text was added to the legend of Fig. 2 [pg 32, line 5] to bring attention to the point that these data are from mice exposed to P. mel. or E. coli LPS in the absence of S. pneumoniae infection, which is also highlighted in the Results section discussing these data [pg 6, lines 13-14]. We also added headers to all serum and flow cytometry data in Fig. 1, Fig. 2, Fig. 3, Fig. 6, and Fig. 8 (and associated Supplementary Figures) denoting whether the data are from infected or uninfected mice to provide additional clarity.
Further analyses of BAL fluid showed a panel of innate immune cytokines (e.g., increase in IL-1α, IFNγ, MCP1, IL6, MIP-2) and the recruitment of neutrophils to the lungs after P. melaninogenica exposure (Ly6G+ cells). In contrast to E. Coli, P. melaninogenica also showed a stronger activation of the neutrophils (Ly6G+ / TNFα+ cells). Monocytes were recruited to the lungs by both strains (Ly6G-/ Ly6C+ cells) and activated (Ly6G-/ Ly6C+ / TNFα+ cells). This overall suggests a ''selective' effect of P. melaninogenica on neutrophils, which potentially mediates the immune protection. When Ly6G+ cells or TNFα from Ly6G+ cells are deleted, the protective effect on bacterial burden ceases.
Investigations into the host and bacterial requirements for P. melaninogenica-induced neutrophil TNFα production showed the importance of TLR2 signalling for this response (inhibition of TLRs). Further, TNFα induction was significantly reduced when heat-killed P. melaninogenica was treated with lipase to digest bacterial lipoproteins in the cell membrane, which are bacterial TLR2 ligands. However, of interest is that additional factors appear to be required to mediate the protective TLR2-lipoprotein signalling effect as neither Pam3SK4 (TLR2 agonist) nor purified P. melaninogenica lipoproteins reduced S. pneumoniae burdens in the lung (Fig. 4f). TLR2-/-mice showed that TLR2 is required for P. melaninogenica-induced neutrophilic activation and TNFα secretion. In the co-infection model, neutrophil recruitment was lost in P. melaninogenica-exposed TLR2 -/-mice infected with S. pneumoniae, while the recruitment of inflammatory monocytes was maintained. This is an important finding that should be added to the figures.
We moved the data showing retention of inflammatory monocyte recruitment in P. melaninogenica-exposed Tlr2-/-mice infected with S. pneumoniae from a Supplementary Fig. to  Fig. 6e, as recommended.
The effect of IL10 in the regulation of protective effects of TNFα Using purified neutrophils, P. melaninogenica induced the secretion of IL-10 in a dosedependent manner, which could regulate TNFα production. Compared to the single infections, co-infection of bone marrow derived neutrophils with P. melaninogenica and S. pneumoniae shows significantly reduced TNFα release. This is also shown as a systemic response in Prevotella-exposed mice. This is not in Fig. 1g, but in fig 7?   Fig. 1g [now Fig. 1e] first shows serum TNF-alpha and IL-10 in WT mice exposed to P. melaninogenica and infected with S. pneumoniae. In Fig. 7 (now Fig. 8c), we add to these data by showing serum TNF-alpha and IL-10 in WT vs. Il10-/-mice. We moved the serum IL-10 data for this Figure to Supplementary Fig. 7c, as we agree this merely confirms loss of serum IL-10